Chemopreventive Task of Capsaicin against Benzo(a)pyrene‐induced Lung Cancer in Swiss Albino Mice

Abstract: A voluminous number of evidence suggests that an increased consumption of fruits and vegetables is a relatively easy and practical strategy to reduce significantly the incidence of cancer. The present study is an effort to identify the chemopreventive role of alkaloid capsaicin against benzo(a)pyrene‐induced lung cancer in Swiss albino mice. Benzo(a)pyrene‐induced lung cancer‐bearing animals showed abnormal changes in body weight, lung weight, tumour incidence and alterations in the activities of marker enzymes adenosine deaminase, aryl hydrocarbon hydroxylase, γ‐glutamyl transpeptidase, 5′‐nucleotidase and lactate dehydrogenase. On capsaicin pre‐co‐treatment, all the above alterations were returned to near normal. Immunohistochemical analysis of proliferating cell nuclear antigen together with lung histological examination further supported our biochemical findings that demonstrated the chemoprotective role of capsaicin against benzo(a)pyrene‐induced experimental lung cancer.     

Benzo(a)pyrene increases phosphorylation of p53 at serine 392 in relation to p53 induction and cell death in MCF-7 cells

Although benzo(a)pyrene (BP) induces apoptosis in vitro in murine Hepa1c1c7 cells and in vivo indications of apoptosis in rat lung exist, related cellular mechanisms in human cells are not known. p53 protein participates in several apoptotic processes. We found that BP induces cell death in human MCF-7 breast adenocarcinoma cells at 48 and 72 h but not in human A549 lung carcinoma cells. BP did not induce measurable caspase-3-like protease activity or internucleosomal DNA fragmentation in either cell types. However, procaspase-7 cleavage in MCF-7 cells by BP-treatment indicates activation of caspase-7 meaning that apoptosis is most likely involved in BP-induced MCF-7 cell death. BP-7,8-dihydrodiol-9,10-epoxide (BPDE)–DNA adducts and level of p53 protein increased dose-dependently, but more extensively in MCF-7 cells. Phosphorylation of p53 protein at serines 15, 20, 46 and 392 increased in MCF-7 cells. Increase in phosphorylation at serine 392 was clear already at 24 h by 1 μM concentration of BP. Increase of phosphorylation at other sites occurred only with higher concentrations or at later time points in relation to the increase of p53 protein. These results suggest that serine 392 phosphorylation is the first stabilizing event of p53 associated with BP exposure and subsequent cell death in MCF-7 cells.     

The Nrf2‐ARE pathway is associated with Schisandrin b attenuating benzo(a)pyrene‐Induced HTR cells damages in vitro

As is ubiquitous in the environmental sources, benzo(a)pyrene (BaP) has been reported to induce reprotoxicity in previous studies. Toxicity to trophoblast cells may be one key factor, but evidences were absent. We speculated that BaP can induce cytotoxicity in human trophoblast HTR‐8/SVneo (HTR) cells, and Schisandrin B (Sch B) as a potential protector can inhibit the cytotoxicity. MTS assay identified that BaP induced HTR cells death while Sch B played a cytoprotective role. And after Nrf2 interference, the ability of Sch B‐induced cytoprotection was declined. Furthermore, PCR, western blot, ELISA, and SOD assays were found that Sch B significantly increased the mRNA and protein expression of Nrf2, HO1, NQO1, and SOD in the Nrf2‐ARE pathway, and the extents of increase were declined after Nrf2 interference. These results demonstrated that the Nrf2‐ARE pathway plays an important role in Sch B attenuating BaP‐induced HTR cells damages in vitro. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1439–1449, 2016.     

Benzo[a]pyrene induced p53‐mediated cell cycle arrest,DNA repair,and apoptosis pathways in Chinese rare minnow (Gobiocypris rarus)

The p53 pathways play an important role in carcinogenesis. In mammals, p53 and p53 target genes have been extensively studied, but little is known about their functions and regulation in fish. In this study, the cDNA fragments of p53 network genes, including p53, p21, mdm2, gadd45α, gadd45β, igfbp‐3, and bax, were cloned from Chinese rare minnow (Gobiocypris rarus). These genes displayed high amino acid sequence identities with their zebrafish orthologs. The mRNA levels of p53 network genes and pathological changes in the liver were determined after adult rare minnow were exposed to 0.4, 2, and 10 µg/L of benzo[a]pyrene (BaP) for 28 days. The results showed that p53, p21, mdm2, gadd45α, and bax mRNA expressions in the livers from males and females were significantly upregulated compared with those of the controls (p < 0.05), but gadd45β and igfbp‐3 expression was not significantly changed. Microphotographs revealed enlargement of the cell nuclei and cellular degeneration in males, while atrophy and vacuolization of hepatocytes were observed in females (10 µg/L). These results suggested that BaP induced liver DNA repair and apoptosis pathways and caused adverse pathological changes in rare minnow. The strongly responsive p53 network genes in the livers suggest that rare minnow is suitable as an experimental fish to screen environmental carcinogens. In addition, the p53 network genes in rare minnow could feasibly be used to identify the mechanism of environmental carcinogenesis. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 979–988, 2017.     

Detection of benzo[a]pyrene‐induced immunotoxicity in orange spotted grouper (Epinephelus coioides)

This study aimed to investigate the effects of benzo[a]pyrene (BaP) on immune status of orange spotted grouper (Epinephelus coioides). Fish were injected with 2, 20 and 35 mg/kg‐bw of BaP and were kept under laboratory conditions for 14 days. Blood samples were taken at days 1, 4, 7, and 14 and changes in total WBC and RBC, phagocytosis, lysozyme activity, lysosomal membrane stability, immunoglobulin M (IgM) level and antibacterial activity were evaluated. Also BaP bioaccumulation in fish muscle was measured. BaP concentration in the muscle of treated fish reached a maximum level after 4 days (P < 0.05). Exposure of fish to BaP resulted in a significant decrease of total RBC and WBC, lysozyme activity, lysosomal membrane stability, IgM level and antibacterial activity after 4 days and phagocytosis after 7 days of the experiment (P < 0.05). Totally, the results revealed BaP ability to suppress the fish immune function. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 329–338, 2016.     

Benzo[a]pyrene‐decreased gap junctional intercellular communication via calcium/calmodulin signaling increases apoptosis in TM4 cells

The mechanism of male reproductive toxicity induced by benzo[a]pyrene (BaP) is poorly understood. Gap junctional intercellular communication (GJIC) is known to play a critical role in maintaining spermatogenesis. The aim of the present study was to determine the toxic effects of BaP in Sertoli cells, and to explore the possibility and potential mechanisms of BaP‐induced changes in the level of GJIC, and the relationship between GJIC and BaP‐induced apoptosis. We treated mouse Sertoli cell lines (TM4) with different concentrations (0.1–100 μm ) of BaP for 1–48 hours, and found that GJIC exhibited a dose‐ and time‐dependent downregulation. Treatment with 10 μm BaP increased apoptosis, intracellular Ca²⁺ level ([Ca²⁺]_i) and calmodulin (CaM) protein expression, and decreased the protein level of connexin 43 (Cx43) (also known as gap junction α‐1 protein [GJA1]) in TM4 cells. However, BaP had no effect on the phosphorylation of Cx43 at Ser279/282, Ser255, Ser368 or Ser262. Downregulation of [Ca²⁺]_i by BAPTA‐AM significantly attenuated the BaP‐induced GJIC suppression, Cx43 protein decrease and CaM protein increase. Interestingly, inhibition of CaM expression by W7 partially recovered BaP‐induced GJIC inhibition, but had no effect on BaP‐induced Cx43 protein decrease. Pretreatment with the GJIC activator retinoic acid significantly mitigated BaP‐induced apoptosis. In conclusion, these results suggest that BaP can decrease GJIC via Ca²⁺/CaM signaling, and that BaP‐induced GJIC suppression increases apoptosis in TM4 cells.     

Protective effect of Diosmin against benzo(a)pyrene‐induced lung injury in Swiss Albino Mice

Diosmin, a naturally occurring flavonoid commonly present in citrus fruit, is known to exhibit anti‐inflammatory, antimutagenic, antioxidant, and free radical scavenging as well as blood lipid lowering activities among others. Diosmin has also been used for the treatment of various diseases including diabetes mellitus and Alzheimer's disease. Our study explores the role of Diosmin in pulmonary toxicity (lung injury) induced by environmental contaminant benzo(a)pyrene [B(a)P]. Swiss Albino Mice (SAM) were administered with either Diosmin 100 or 200 mg/kg body weight daily for 14 days and then challenged with a single dose of B(a)P. On the 15th day, animals were sacrificed; lung tissues and blood were collected for molecular analysis. B(a)P administration in mice induced the thickening of lung epithelium, damaged alveolar architecture, and promoted inflammatory cell infiltration in the lung tissues. Also, B[a]P significantly increased the expression of NF‐kB, COX‐2, IL‐6, Bax, cleaved caspase 3, and cleaved PARP proteins and decreased antioxidant enzyme levels. Diosmin‐100 and Diosmin‐200 significantly attenuated the damage to lung epithelium, alveolar architecture, and reduced inflammatory cell infiltration in the lung tissues of mice. Diosmin significantly (P < .05) attenuated the levels of oxidative stress markers: lactate dehydrogenase and xanthine oxidase. A decrease in expression of NF‐kB, COX‐2, IL‐6, Bax, cleaved caspase 3, and cleaved PARP proteins in mice was challenged with B[a]P. Diosmin thus could be a promising therapeutic adjuvant against B[a]P‐induced oxidative stress and lung damage.     

Searching for Dual Inhibitors of the MDM2‐p53 and MDMX‐p53 Protein–Protein Interaction by a Scaffold‐Hopping Approach

Two libraries of substituted benzimidazoles were designed using a ‘scaffold‐hopping’ approach based on reported MDM2‐p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X‐ray structure. Benzimidazole libraries were prepared using an efficient solution‐phase approach and screened for inhibition of the MDM2‐p53 and MDMX‐p53 protein–protein interactions. Key examples showed inhibitory activity against both targets.     

Involvement of P450 1A1 in Benzo(a)Pyrene but Not in Benzo(a)Pyrene-7,8-Dihydrodiol Activation by 3-Methylcholanthrene-Induced Mouse Liver Microsomes

Abstract: Synchronous fluorescence spetrophotometry for benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE)-DNA adducts was used to study the activation pathway of benzo(a)pyrene in C57BL/6 mice. Benzo(a)pyrene but not benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced mouse liver microsomes was inhibited by a monoclonal antibody (Mab 1–7–1) against CYP1A1/2 suggesting that 1A1 probably takes part in the first P450 reaction. However, aryl hydrocarbon hydroxylase activity, a classical measure of benzo(a)pyrene metabolism, was not inhibited by the same concentration of Mab 1–7–1. None of the other antibodies used, detecting 2A, 2B, 2C or 2E subfamilies, inhibited the adduct formation. Troleandomycin and gestodene, chemical inhibitors of human 3A4, inhibited benzo(a)pyrene-7,8-diol activation by 3-methylcholanthrene-induced microsomes to some extent only in high concentrations. Although liver microsomes from 3-methylcholanthrene-induced mice catalyzed the formation of BPDE-DNA in vitro clearly more than uninduced microsomes, 3-methylcholanthrene pretreatment in vivo decreased the adduct formation in benzo(a)pyrene-treated mice. These results emphasize the significance of detoxicating and DNA-repairing pathways in vivo. Finally, synchronous fluorescence spectrophotometry for BPDE-DNA measures the end-point of the three-step activation pathway while aryl hydrocarbon hydroxylase measures a one-step hydroxylation. Thus, these methods should be used rather as corroborative than mutually exclusive assays.     

Dynamic Epigenetic Changes Involved in Testicular Toxicity Induced by Di‐2‐(Ethylhexyl) Phthalate in Mice

Abstract: The aim of this study was to analyse epigenetic (specifically, DNA methylation) change in testes induced by maternal exposure to di‐2‐(ethylhexyl) phthalate (DEHP) in mice. Testicular dysgenesis syndrome was induced in foetuses by maternal exposure to DEHP. High‐performance liquid chromatography was performed to analyse DNA methylation status, and expression levels of the DNA methyltransferases were examined by quantitative real‐time polymerase chain reaction and western blotting. DEHP significantly had more than 10% relative increase in the global DNA methylation and also increased DNA methyltransferases’ expression. Changes in DNA methylation may play an important role in abnormal testicular function caused by environmental factors such as maternal exposure to DEHP, which may be one possible mechanism of DEHP‐mediated testicular toxicity.     

Oncogenicity evaluation of resveratrol in p53(+/-) (p53 knockout) mice.

A six-month study was conducted in p53(+/-) mice to evaluate the possible oncogenicity of resveratrol (3,5,4'-trihydroxy-trans-stilbene), a cancer chemopreventive agent present in grapes and other foods. p53(+/-) mice (25/sex/group) received daily gavage exposure to vehicle only (negative control), resveratrol doses of 1000, 2000, or 4000 mg/kg/day, or p-cresidine (400 mg/kg/day; positive control). No mortality was seen in mice receiving the low dose of resveratrol. However, the mid and high doses induced mortality associated with impaction of the test article in the gastrointestinal tract. Resveratrol had no effect on body weight, food consumption, or clinical signs in surviving mice in any dose group, but induced dose-related increases in liver weight and serum cholesterol in both sexes. Mild anemia was seen in male mice at the high dose only; hematologic effects were not seen in females. Histopathology identified the kidney (hydronephrosis) and urinary bladder (epithelial hyperplasia) as target tissues for resveratrol toxicity. The incidences of both benign and malignant tumors in mice exposed to resveratrol were comparable to those in vehicle controls. By contrast, the positive control article, p-cresidine, induced urinary bladder cancer in both sexes. When administered to p53(+/-) mice at its maximum tolerated dose, resveratrol demonstrates no evidence of oncogenicity.     

宫颈癌p53外显子7突变与p53蛋白高表达的关系

目的　探讨宫颈癌 p5 3外显子 7突变与 p5 3蛋白高表达的关系。方法　采用免疫组织化学、聚合酶链反应 ( PCR)、限制性酶解片段长度多态性 ( RFL P)分析等方法对 49例宫颈癌组织石蜡包埋标本中 p5 3外显子 7的突变与 p5 3蛋白表达进行了检测。结果　 p5 3外显子 7的突变率 8.2 % ( 4/ 49)显著低于 p5 3蛋白阳性率49.0 % ( 2 4/ 49) (χ2 =18.0 5 ,P<0 .0 0 1) ;p5 3外显子 7突变不一定 p5 3蛋白阳性。结论　 p5 3外显子 7突变可能是部分宫颈癌变的一个重要因素 ;大部分宫颈癌可能主要由于高危人乳头状瘤病毒 ( HPV)感染后 ,通过 E6 / p5 3蛋白复合物的形成使 p5 3蛋白失活所致     

The Effects of l‐Carnitine Against Cyclophosphamide‐Induced Injuries in Mouse Testis

In order to explore the possibility of l ‐carnitine (LC) as a protector of male fertility in chemotherapy, we observed the damage of cyclophosphamide (CTX) to Sertoli cells and the protective effect of LC on the testis Sertoli cells from such damage in this study. Healthy adult male mice were divided into three groups: chemotherapy group were injected intraperitoneally with the CTX; protective agent group were injected both LC and CTX; control group mice were injected only with isochoric physiological saline; all once a day for 5 days. After 5 days, the mice were, respectively, killed at 24 hr after the last injection. The testis and epididymis were removed. Epididymis was for sperm analysis immediately, and immunohistochemistry, RT‐PCR and Western blot for the assessments of occludin, glial cell‐derived neurotrophic factor (GDNF) and TGF‐β3 mRNA and protein expression. The sperm analysis of epididymis showed that CTX can significantly decrease sperm count and motility; and administration of LC resulted in significant recovery of the sperm count and sperm motility. Compared with control group, the expressions of occludin and GDNF decreased and the expression of TGF‐β3 increased significantly (p < 0.05) in the CTX group. In the LC + CTX group, the expressions of occludin and GDNF were higher than those of the CTX group and similar to those of the control group; the TGF‐β3 expression was lower (p < 0.05) than that of the CTX group and similar to that of the control group. The results of this study showed that CTX could damage the spermatogenesis and reduce the expression of occludin and GDNF, and increase the expression of TGF‐β3 in testis of mouse, which indicates CTX's damage or efficacy to testis Sertoli cells. LC could protect the Sertoli cells of testis from these damages caused by CTX, and promote or protect the spermatogenesis. In conclusion, this study provides meaningful information about the possible damage to male fertility by chemotherapeutics and potential of LC in the protection of male fertility during chemotherapy.     

p53-Mdm2 inhibitors: patent review (2009 - 2010)

INTRODUCTION: p53 plays a central role in protecting the integrity of the genome. Its activity is ubiquitously lost in cancers, either by inactivation of its protein (p53 pathway) or by mutation in the p53 gene, thereby indicating its importance in understanding cancer and as a therapeutic target. Activated p53 is known to induce cell cycle arrest thereby leading to apoptosis and has been the subject of intensive research in the area of medicinal chemistry. Efforts are in progress to synthesize a variety of scaffolds that could inhibit the p53-Mdm2 interaction by binding to Mdm2 in the region where p53 is likely to bind. These molecules have the potential to be developed as anticancer drug candidates and have been largely explored by both academia and industry. Interestingly, some of these molecules are in the early stage of clinical trials. AREAS COVERED: Areas covered in this review include patents relating to p53-Mdm2 inhibitors during the time period 2009 - 2010. The focus of the review was on small-molecule inhibitors. EXPERT OPINION: Inducing apoptosis in cancerous cells by the activation of p53 is an area that is being actively explored. There are strong indications that it could become a therapeutic method for the treatment of cancer. As a result, extensive research is being performed by both academia and industry. It is observed that small molecules that are present in early clinical trials are expected to be developed as potential drugs for cancer therapy.     

Association of p38MAPK‐p53‐Fas aggregation in S‐allyl cysteine mediated regulation of hepatocarcinoma

Bioactive components of dietary phytochemicals have been reported to possess antitumor activities. Evidences suggested key role of stress responsive p38MAPK in the induction of nutraceuticals mediated apoptosis in hepatocellular carcinoma (HCC). Current study demonstrated detailed molecular bagatelle associated with p38 MAPK mediated effective suppression of cell growth both in HepG2 and chemically induced liver carcinoma after S‐allyl cysteine (SAC) treatment. SAC promoted p38MAPK activity responsible for p53 phosphorylation, its stabilization followed by nuclear translocation leading to induction in expression and oligomerization of Fas protein. Distinctive p38MAPK‐p53 axis dependent Fas‐FasL‐FADD mediated caspase activities along with perturbed cell cycling became normalized with continuation of SAC treatment for another month to diethylnitrosamine induced liver carcinoma. Co‐treatment with SB203580, the p38MAPK inhibitor, prevented pro‐apoptotic effect of SAC by altering p53 phosphorylation and death inducing signaling complex conformation in HepG2 and induced HCC. Collectively study suggested significant contribution of p38MAPK‐p53‐DISC‐Caspase pathway in the regulation of anti‐neoplastic activity of SAC against HCC.     

p53–Mdm2 inhibitors: patent review (2009 – 2010)

Introduction: p53 plays a central role in protecting the integrity of the genome. Its activity is ubiquitously lost in cancers, either by inactivation of its protein (p53 pathway) or by mutation in the p53 gene, thereby indicating its importance in understanding cancer and as a therapeutic target. Activated p53 is known to induce cell cycle arrest thereby leading to apoptosis and has been the subject of intensive research in the area of medicinal chemistry. Efforts are in progress to synthesize a variety of scaffolds that could inhibit the p53–Mdm2 interaction by binding to Mdm2 in the region where p53 is likely to bind. These molecules have the potential to be developed as anticancer drug candidates and have been largely explored by both academia and industry. Interestingly, some of these molecules are in the early stage of clinical trials.

Areas covered: Areas covered in this review include patents relating to p53–Mdm2 inhibitors during the time period 2009 – 2010. The focus of the review was on small-molecule inhibitors.

Expert opinion: Inducing apoptosis in cancerous cells by the activation of p53 is an area that is being actively explored. There are strong indications that it could become a therapeutic method for the treatment of cancer. As a result, extensive research is being performed by both academia and industry. It is observed that small molecules that are present in early clinical trials are expected to be developed as potential drugs for cancer therapy.